1. Field of the Invention
The present invention relates to the enhanced production and accumulation of terpenes in plants via the expression of fusion proteins comprising various combinations of geranyl diphosphate synthase large and small subunits with limonene synthase. The present invention also relates to engineering of oilseed plants, exemplified by camelina, to accumulate monoterpene and sesquiterpene hydrocarbons, exemplified herein by the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon 5-epi-aristolochene. This establishes a framework for the rapid engineering of oilseed crop production platforms for terpene-based biofuels.
2. Description of Related Art
Jet fuel is a mixture of many different hydrocarbons. Modern analytical techniques indicate that there may be a thousand or more. The range of their sizes (carbon numbers) is restricted by specific physical requirements of a specific jet fuel product. Kerosine-type jet fuel has a carbon number distribution between about 8 and 16 carbons. Most of the hydrocarbons in jet fuel are members of the paraffin, naphthene and aromatic classes. The compounds that boil near the middle of the kerosine-type jet fuel boiling-range are C10 aromatics, C11 naphthenes, and C12 waxes. Given the decline in oil based natural resources, and potential for environmental disasters associated with oil extraction and transport, there is renewed interest in identifying renewable sources of jet fuels and related industrial hydrocarbon based products.
Plants synthesize a wide repertoire of cyclic and linear low molecular weight hydrocarbon compounds, which have the potential to be readily converted into jet fuel and industrial solvents. For example, the cyclic monoterpene, limonene, (4S)-1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene) occurs naturally in various ethereal oils, particularly oils of lemon, orange, caraway, dill and bergamot, and is a valuable industrial chemical. Some limonene is prepared by extraction from plants of the mint family, a large quantity is obtained from citrus oils, which are typically 80-90% limonene, and some is obtained from pine oil. It is also synthesized chemically and finds use as a solvent and cleaning agent (in the manufacture of synthetic pine oil), as an expectorant, as a wetting and dispersing agent, as a monomer in the manufacture of various polymeric resins, as a flavorant and a precursor in the synthesis of the flavorant carvone, and as a polymerization inhibitor in storage of the tetrafluoreoethylene monomer used in the manufacture of polytetrafluoroethylene (PTFE).
In principal the introduction of relatively few low molecular weight metabolite biosynthetic genes into a heterologous host such as an oilseed plant, or an alga could result in the production and accumulation of a variety of hydrocarbons that could serve as chemical precursors to wide range of industrial aromatic hydrocarbons including, C10 aromatics, C11 aromatics which are widely used as solvents and fuels.
In planta, C-10 terpenes (monoterpenes) are synthesized in plastids of specialized gland cells (Turner et al., (1999). Plant Physiology 120: 879-886) from precursors derived via the non-mevalonate pathway from pyruvate and glyceraldehyde-3-phosphate (Rohdich et al., Current Opinion in Chemical Biology 5: 535-540). C-15 terpenes (sesquiterpenes) are synthesized in the cytosol via the mevalonate pathway from acetyl-CoA (Chappell, J (2004) Trends in Plant Science. 9: 266-269). The volatile products of mono- and sesquiterpene biosynthesis in most plants are either secreted into specialized storage cavities or are released to the atmosphere.
The first committed step of monoterpene (see FIG. 2) biosynthesis is mediated Geranyl diphosphate synthase (GDS) which catalyzes the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to form GPP, the immediate acyclic precursor of monoterpenes. GPP is converted to (−)-4S-limonene by the catalytic action of (−)-4S-limonene synthase (cyclase), which represents the primary precursor of various monoterpenes including its downstream metabolites (−)-trans-carveol and carvone; as well as the precursor of S-linalool. See FIG. 1; Wise et al. (1997) In “Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2” (Cane, D. E., ed.), Elsevier Science, Oxford (1998).
Both GPP synthase and 4S-limonene synthase has been isolated from several plant sources, including grape, geranium, sage (Croteau et al. (1989) Arch. Biochem. Biophys. 271:524-535; Heide et al. (1989) Arch. Biochem. Biophys. 273:331-338; Suga et al. (1991) Phytochemistry 30:1757-1761; Clastre et al. (1993) Plant Physiol. 102:205-211); and spearmint (Colby et al., (1993) J. Biol. Chem. 268(31) 23016-23024) and various cDNA clones are publicly available.
Despite the availability of these clones, previous systems for the production of Limonene and other downstream metabolites of related monoterpenes have primarily focused on the use of such systems for insect control. (See for example, U.S. Pat. No. 6,291,745). In this case, the emphasis was on producing sufficient amounts Limonene in plant tissues such as root to provide effective insect resistance, which was reported to be in the range of 200 ppm, rather than the high level stable production and stable accumulation of mg quantities of terpenes in plant seeds. Camelina sativa is an oilseed plant that has been little exploited in agriculture. It is similar in appearance to oilseed rape and similar in genetic characteristics to Arabidopsis thaliana. As Arabidopsis, it can be readily transformed by floral dip. Camelina is not a foodstuff plant and grows on marginal lands (e.g. Montana) that are generally considered unsuitable for large scale food production. Camelina is being investigated as a winter crop for southern Missouri and could potentially be double-cropped with soy. These characteristics make Camelina an ideal candidate plant to be developed as a chemical factory, particularly if high level production and accumulation of chemicals can be demonstrated in seeds. It is believed, however that the successful large scale biosynthesis and production of terpenes in Camelina seed has not been previously reported.
The current invention is based, at least in part, on the surprising discovery that the over expression of fusion proteins comprising either the GPP synthase large and small subunits, and limonene synthase, or one or more of these subunits fused to limonene synthase, in Camelina seeds results in the high level production and stable accumulation of various terpenes within the seeds. The present invention also surprisingly demonstrates that plants, in particular oil seed crops, can produce and accumulate monoterpene and sesquiterpene hydrocarbons in seeds. The resulting transgenic plants provide for the first time a viable approach for the large scale commercial production of commercially important terpenes in plants, with the potential to directly provide a renewable source of aromatic hydrocarbons, suitable for use for the production of jet fuel, organic solvents, plastics and high value industrial raw materials.